Summary: To advance our long-term goal of understanding the regulation of immunoglobulin isotype switching in human B lymphocytes, we have been studying three enhancer regions lying downstream of the human immunoglobulin C-alpha genes to determine how these regions integrate signals from the B cell milieu to regulate Ig expression in conjunction with Ig gene promoters and other potential regulatory regions such as insulators. We have stably transfected several cell lines with GFP constructs linked to various versions of the enhancer region to test for position-independent copy-number-dependent expression indicative of LCR (Locus Control Region) function of the enhancer complex; clones are currently being analyzed. We have recently been studying the chromatin context of this DNA locus using chromatin immunoprecipitation (ChIP) assays to identify the boundaries of a putative transcriptional activation domain surrounding the immunoglobulin heavy chain locus in human and the homologous murine DNA; we are assessing several amplicons throughout the murine heavy chain locus and downstream for in vivo binding to modified histone proteins or transcription factors. We are also looking for regulatory boundaries using an enhancer blocking assay in which candidate insulator DNA fragments are inserted between a promoter and enhancer of a neomycin resistance gene, which is then transfected into a cell line. The number of neomycin-resistant colonies reflects the extent of expression of the neomycin resistance gene; a classical insulator should decrease expression by shielding the promoter from the influence of the enhancer. We have obtained preliminary evidence for an insulator downstream of the human heavy chain locus, and are further studying this region and fragments downstream of the murine IgH locus. In a collaborative project with Robert Crouch (NICHD) and Tasuku Honjo (Kyoto University) we have been exploring the mechanism of immunoglobulin isotype switching by asking whether the switching observed in CH12F3, a murine B cell line, would be affected by over-expression of the enzyme RNase H, which cleaves RNA bound to DNA. Using a tetracycline-regulatable RNAse H gene, we have found that over-expression of this enzyme leads to a distinct population of cells that express both IgM and IgA on their surface, but that the cells return to IgM-only expression when RNase H expression is down-regulated. In addition, low levels of RNase H expression lead to an increase in cells that are positive for surface IgA only and that secrete IgA. In related work using the same cell line we have detected DNA breaks in the switch region of the alpha locus which are dependent on incubating the cells with IL4, TGF-beta and CD40 Ligand, conditions that cause switching to this locus; these breaks likely represent intermediates in the switch recombination.